The specific degradation of proteins by the 26S proteasome is a key mechanism in cell cycle control. Proteins to be degraded are modified with a ubiquitin chain, which is catalyzed by ubiquitin ligases. By contrast, specific deubiquitinating enzymes can remove ubiquitin chains and thereby rescue proteins from degradation. The misregulation of ubiquitination and deubiquitination can cause aberrant proliferation and cancer. It is our long term goal to understand how the dynamic modification of proteins with ubiquitin controls cell cycle progression, and how it is misregulated in cancer. To address these questions, we focus on the ubiquitin ligase Anaphase-Promoting Complex/Cyclosome (APC/C). The APC/C is essential for proliferation in all eukaryotes. It orchestrates progression through mitosis by promoting the degradation of cell cycle regulators in a highly conserved sequence, which is referred to as substrate ordering. The APC/C achieves substrate ordering by catalyzing the formation of ubiquitin chains with different degrees of processivity. The more processive the ubiquitination of an APC/C-substrate, the earlier it is degraded in mitosis. The activity of the APC/C and the timing of degradation of APC/C-substrates during mitosis are regulated by specific deubiquitinating enzymes. Therefore, a tight interplay between ubiquitin chain formation by the APC/C and deubiquitination by specific deubiquitinating enzymes accounts for the controlled progression of cells through mitosis. In specific aim (1) of this application, we will dissect the mechanisms that underlie ubiquitin chain formation by the APC/C, and thus, substrate ordering. In specific aim (2), we will reconstitute deubiquitinating enzymes that regulate APC/C-activity and substrate degradation. Dissecting the substrate specificity and regulation of these enzymes will allow us to determine the importance of deubiquitination for cell cycle regulation and could lead to the identification of new cell cycle regulators. Finally, in specific aim (3), we will test the hypothesis that the co-regulation of ubiquitin ligases and deubiquitinating enzymes can account for multiple layers of cell cycle regulation, including the transient inactivation of cell cycle regulators, in the absence of proteasomal degradation. We will use a complementary approach of mechanistic studies in vitro and live-cell analysis in vivo to address these important questions. Our experiments will shed light onto fundamental aspects of cell cycle control and could identify new cell cycle regulators. Importantly, the misregulation of the APC/C during early mitosis can underlie the resistance of cancer cells against treatment with the chemotherapeutic taxol. Our reconstitution of key cell cycle reactions in vitro could lay the groundwork for screens aimed at identifying small molecules that specifically inhibit APC/C and restore the responsiveness of cancer cells to taxol treatment.